DESCRIPTION: The major objective of this project is to elucidate the functions of heat shock proteins from the HSP70 family in viral pathogenesis. The cellular HSP70s are vital in stress responses, protein folding, translocation, and assembly, acting via protein-protein interactions modulated by an ATPase activity. Divers RNA and DNA viruses, including human pathogens, recruit HSP70s at various stages of their life cycle, or induce HSP70s following viral infection. However, the general role of protein chaperones in virus-host interactions is unknown. Beet yellows closterovirus (BYV) employed in this project encodes a HSP70-homolog that is essential for virus translocation from cell to cell. Use of BYV as a model provides unprecedented opportunity to directly probe the functions and mechanisms of HSP70h action in viral pathogenesis. Three specific aims of this project will address structure/function relations, subcellular distribution, and regulation of HSP70h expression. A. Functional dissection of HSP70h. Functionally important structural elements, role of the ATPase activity, and specialization of the HSP70h domains will be characterized using reverse genetic and biochemical approaches. Structural conservation among cellular and viral HSP70s will be used to design mutations that will be introduced into the full-length BYV cDNA. The availability of BYV tagged by GUS or GFP reporters will facilitate functional study of mutant phenotypes. B. Protein partners of HSP70h. Since all cellular HSP70s operate via protein-protein interactions, identification of target protein of HSP70h will provide important clues as to the mechanism of HSP70h action. Partner proteins of HSP70h will be identified using affinity chromatography, immunohistochemistry, and in vivo assays provided by the yeast two-hybrid system. C. Subcellular distribution and regulation of HSP70h. Localization of the HSP70h in infected cells and tissues will be examined by an immunohistochemical approach. In addition, HSP70h-GFP fusions produced by modified viruses will be utilized to reveal HSP70h distribution in live cells. The transcriptional and translational regulation of HSP70h will be studied in protoplasts transfection experiments using recombinant BYV expressing a reporter gene under control of the HSP70h promoter.